Tissue inplant systems and methods for sustaining viable high cell densities within a host

ABSTRACT

Implant assemblies and methodologies provide immuno-protection for implanted allografts, xenografts, and isografts. The assemblies and methodologies establish a boundary between the host and the implanted cells. The boundary has a pore size, an ultimate strength, and a metabolic transit value that assures the survival of the cells during the critical ischemic period and afterward. The boundary allows the fabrication and clinical use of implant assemblies and methodologies that can carry enough cells to be of therapeutic value to the host, yet occupy a relatively small, compact area within the host.

RELATED APPLICATION

This application is a continuation in part of copending U.S. applicationSer. No. 735,401 entitled "Close Vascularization Implant Material" filedJul. 24, 1991, now abandoned.

FIELD OF THE INVENTIONS

The inventions relate to systems and methods for implanting living cellswithin a host.

BACKGROUND OF THE INVENTIONS

For several years, researchers have been trying to surgically implantliving cells in a host to treat various cell and molecular deficiencydiseases. In theory, the implanted cells will generate biologicalproducts that the host, because of disease or injury, cannot produce foritself. For example, the implant assembly can contain pancreatic cells(clusters of which are called "islets"), which generate insulin that adiabetic host lacks.

Yet, in practice, conventional implant assemblies and methodologiesusually fail to keep the implanted cells alive long enough to providethe intended therapeutic benefit. For example, pancreatic cellsimplanted for the treatment of diabetes usually die or becomedysfunctional within a few days or weeks after implantation.

For a period after implantation, the region of the host tissue next tothe implant assembly can be characterized as ischemic. "Ischemic" meansthat there is not a sufficient flow of blood in the tissue regionclosely surrounding the implant assembly. Usually, this ischemiccondition exists during the first two weeks of implantation. Mostimplanted cells fail to live through this period.

During the ischemic period, a foreign body capsule forms around theimplanted cells. The capsule consists of flattened macrophages, foreignbody giant cells, and fibroblasts. Conventional hypotheses blame theforeign body capsule for causing implanted cells to die or becomedysfunctional during the ischemic period.

The inventors have discovered that these widely held hypotheses arewrong. The inventors have discovered that the cells do not die becauseof the intervention of the foreign body capsule. Instead, the cells diebecause conventional implant assemblies and methodologies themselveslack the innate capacity to support the implanted cells' ongoing lifeprocesses during the critical ischemic period, when the host's vascularstructures are not nearby. Because of this, the implanted cells perishbefore the host can grow new vascular structures close enough to sustainthem.

When implanted cells die during the ischemic period, a classical foreignbody capsule inevitably forms around the implant. The persistentpresence of this capsule led previous researchers to the falseconclusion that the host's foreign body reaction was the cause ofimplanted cell death, rather than its result.

The invention corrects these and other problems in existing implantassemblies and methodologies.

Many previous implant assemblies have also failed to be useful in aclinical setting, because they cannot be practically implanted andtolerated by the host without danger or discomfort.

For example, an implant assembly that housed cells within hollow fiberswas recently used by CytoTherapeutics to successfully treat diabetes inrats. The assembly consisted of 7 fibers, each being 2 cm long and 0.073cm in diameter. The pancreatic cells were present within the fibers at adensity of about 25,000 cells per cm³. For this assembly to beclinically useful for the treatment of diabetes in humans, it would haveto contain at least about 250,000 pancreatic islets (each islet containsabout 1000 cells). This means that, to hold enough pancreatic cells totreat human diabetes, the assembly would have to be about 117 feet long.This makes the assembly unusable for clinical use in humans.

Recently, cells have also been encapsulated in tiny hydrogel vessels,called microcapsules. These tiny vessels cannot be implanted within thehost's soft tissues, because they lack the physical strength towithstand the physiological stresses normally encountered close to thehost tissue. Instead, the microcapsules are suspended in a free floatingstate within a solution that is infused into the host's peritonealcavity.

In reality, the microcapsules have only limited clinical application.Not all persons can tolerate their injection free of danger ordiscomfort. Microcapsules are non-adhesive, and they do not stick toorgans. Instead, they settle in large masses at the bottom of theperitoneal cavity. And, if implanted directly within the host's tissue,the microcapsules will rupture, and the contained cells would perish.For these reasons, microcapsules fail to provide a widely usableclinical solution to the problems surrounding the therapeuticimplantation of cells.

The inventions have as an important objective the design of implantassemblies and methodologies that combine effectiveness and practicalityrequired for widespread clinical use.

SUMMARY OF THE INVENTION

To meet these and other objectives, the inventions provide improvedimplant assemblies and methodologies that can carry enough cells to beof therapeutic value to the host, yet occupy a relatively small, compactarea within the host. The implant assemblies and methodologies that theinventions provide also establish an improved boundary between theimplanted cells and the host. The improved boundary sustains theviability of the implanted cells, both before and after the growth ofvascular structures by the host.

To assure the long term survival and functionality of implanted cells,the host must grow new vascular structures to serve them. The inventorshave discovered that an animal host will not naturally provide these newvascular structures. It must be stimulated to do so.

The implant assembly itself must provide this crucial stimulation to thehost. Otherwise, new vascular structures will not form close to theboundary. The implanted cells will die or will not function as expected.

The inventors have found that some cells implanted for therapeuticreasons, like pancreatic islets, naturally secrete angiogenic material."Angiogenic" identifies a type of material that has the characteristicof stimulating the growth of new vascular structures by the host closeto the boundary that separates the implanted cells from the host."Close" means that the vascular structures generally lie within aboutone cell layer away from the boundary, which is usually less than about15 microns.

These angiogenic source cells, if implanted, create their ownstimulation for close neovascular growth. Yet, other cells do notnaturally secrete angiogenic materials. These cells, if implanted alone,will not induce vascularization. If these cells are implanted, theimplant assembly should include a separate angiogenic source for them.

Still, the presence of an angiogenic source does not assure cellsurvival during the ischemic period, before the close vascularstructures form. Even cells that naturally secrete angiogenic materialoften die or become dysfunctional soon into the ischemic period. Theirrelease of angiogenic material stops, too, bringing vascularization to ahalt.

The inventors have discovered that implanted cells perish during theischemic period, because the assemblies housing them lack the intrinsiccapacity to bring in enough nutrients and let out enough wastes tosupport their ongoing metabolic processes when the host's vascularstructures are absent. This capacity will be referred to as "metabolictransit."

It is the lack of sufficient metabolic transit innate in prior implantassemblies and methodologies, and not the formation of the foreign bodycapsule, that causes the implanted cells to expire or becomedysfunctional during the ischemic period. It is the lack of sufficientmetabolic transit by the boundary that stymies the formation of closevascular structures and causes the implant to fail.

The inventors have discovered that an implant assembly will support theongoing metabolic processes of implanted cells during the ischemicperiod, even when a foreign body capsule forms, if the assembly has asufficient metabolic transit value to support these processes in theabsence of close vascular structures. With their metabolic processessupported, the cells survive the ischemic period. When the assemblyincludes implanted angiogenic source cells, thy also release theirangiogenic materials to stimulate new vascular structures. Formation ofthe new vascular structures, in turn, marks the end of the ischemicperiod. A sufficient metabolic transit value sustains and promotes allthese complementary processes.

One aspect of the inventions provides implant assemblies andmethodologies that present an improved boundary between the host tissueand the implanted cells. The boundary is characterized in terms of itspore size; its ultimate physical strength; and its metabolic transitvalue. The metabolic transit value is, in turn, characterized in termsof the permeability and porosity of the boundary.

The pore size and ultimate physical strength characteristics serve toisolate the implant tissue cells from the immune response of the hostduring the ischemic period and afterward. The metabolic transit valueserves to sustain viability of the implanted cells during the ischemicperiod and afterward, even when a foreign body capsule forms.

In a preferred arrangement, the boundary has a surface conformation thatalso supports and fosters the growth of the new vascular structures thatthe improved implant assemblies and methodologies stimulate.

Another aspect of the inventions provides a methodology to derive anduse a therapeutic loading factor to characterize and predict theclinical effectiveness of a given implant assembly for a given celltype. The therapeutic loading factor takes into account the number ofcells that are required to be implanted to achieve the desiredtherapeutic effect; the effective area of the boundary between theimplanted cells and host that the host can be reasonably expected totolerate; and the metabolic transit value needed to sustain cellviability. Using the therapeutic loading factor, a practitioner canprovide an implant assembly that combines the benefits of compact sizewith the ability to sustain the requisite therapeutical number of cells.

The inventions provide implant assemblies and methodologies havingsignificantly improved performance characteristics. The improvedcharacteristics sustain high density cell populations within a compactarea within a host. Assemblies and methodologies that embody thefeatures of the inventions support as many as 8 times more implantedcells in a given volume than prior assemblies and methodologies.

Other features and advantages of the inventions will become apparentupon review of the following specification, drawings, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an implant assembly that embodies thefeatures of the invention being held in the hand of a practitioner;

FIG. 2 is an enlarged perspective view of the implant assembly shown inFIG. 1;

FIG. 3 is an enlarged and exploded perspective view of the implantassembly shown in FIG. 2;

FIG. 4 is a side section view of the implant assembly taken generallyalong line 4--4 in FIG. 2;

FIG. 5 is an enlarged and exploded perspective view of another implantassembly that embodies the features of the invention, showing thepractitioner loading implanted cells into the assembly;

FIG. 6 is an enlarged assembled view of the assembly shown in FIG. 5,before the formation of a peripheral seal;

FIG. 7 is an enlarged view of the assembly shown in FIG. 6, partiallypeeled apart to show the interior;

FIG. 8 is an enlarged assembled view of the assembly shown in FIG. 5after the formation of a peripheral seal;

FIG. 9 is a side section view of a portion of the sealed assembly takengenerally along line 9--9 in FIG. 8;

FIG. 10 is a side section view of the assembly before sealing, takengenerally along line 10--10 in FIG. 6;

FIG. 11 is a perspective view of a lamination slide holding the bottomlayer of the laminated boundary structure that embodies the features ofthe invention;

FIG. 12 is a side section view of the lamination slide taken generallyalong line 12--12 in FIG. 11;

FIG. 13 is a perspective view of several lamination slides laid side byside for the application of adhesive filaments in the process of makingthe laminated boundary structure;

FIG. 14 is a side section view of the laminated boundary structure withits top layer laid over the cement filaments applied in FIG. 13;

FIG. 15 is a side section view of the laminated boundary structureclamped between two lamination slides while the cement filaments cure;

FIG. 16 is a perspective view of individual boundary wall elements beingcut from the laminated structures made following the steps shown inFIGS. 11 to 15;

FIG. 17 is a diagrammatic depiction of an implant assembly that embodiesthe features of the invention after having bee surgically implanted inhost tissue;

FIG. 18 is a diagrammatic depiction of the implant assembly during theischemic period, after about one or two days of implantation, showingthe surrounding wound area filled with exudate;

FIG. 19 is a diagrammatic depiction of the implant assembly after abouttwo weeks of implantation, showing the formation of vascular structuresclose to the boundary, ending the ischemic period;

FIG. 20 is a diagrammatic depiction of a section of the implant assemblyin which the implanted cells have survived the ischemic period, showingthe formation of vascular structures close to the boundary and theresulting alteration of the foreign body capsule;

FIG. 21 is a diagrammatic depiction of a section of the implant assemblyin which the implanted cells have not survived the ischemic period,showing the lack of vascular structures close to the boundary and theresulting intervention of the foreign body capsule;

FIG. 22 is a graph showing the therapeutic loading curve for pancreaticcells derived in accordance with the invention.

Before explaining the preferred embodiments, it is to be understood thatthe inventions are not limited in use to the details of construction ormethodologies there set forth or as illustrated in the drawings. Theinventions are capable of other embodiments and of being practiced andcarried out in various ways.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIGS. 1 to 4 show an implant assembly 10 that embodies the features ofthe invention.

The assembly 10 can carry preselected types of living cells 12 forimplanting within the soft tissue of a host. The implanted cells 12generate biological products that the host, because of disease orinjury, cannot produce for itself.

For example, the implant assembly 10 can carry clusters of pancreaticcells (called "islets"), which generate insulin for release into and useby a diabetic host.

The assembly 10 forms a porous, life sustaining boundary between theimplanted cells 12 and the host. The porous boundary isolates theimplanted cells 12 from attack and destruction by certain biologicalmechanisms of the host. At the same time, the porous boundary associateswith the host's biological system closely enough to transfer nutrientsand wastes in support of the biological processes of the implanted cells12. The porous boundary also transfers the therapeutic productsgenerated by the implanted cells 12 to the host.

In the embodiment shown in FIGS. 1 to 4, the assembly 10 includes ahoop-like housing 11. The housing includes a first hoop element 14 and asecond hoop element 16 that nests within the first hoop element 14. Theassembly 10 also forms a cell chamber 18 within the hoop-like housing11.

The first hoop element 14 has an upstanding cylindrical side wall 20that peripherally defines an open area. First and second centralopenings 22 and 24 lead into the open area. The first central opening 22is smaller than the second central opening 24. This forms an interiorstep or ledge 26 next to the first opening 22.

The second hoop element 16 also has a central opening 28. The secondhoop element 16 has an outer diameter that is slightly greater than theinner diameter of the open area of the first hoop element 14. Theperipheral edge of the second central opening 16 contains a slightchamfer 30 to receive the second hoop element 16. When assembled, thesecond hoop element 16 nests snugly in an interference press fit withinthe open area of the first hoop element 14 (see FIG. 2).

The first hoop element 14 and the second hoop element 16 are made of adurable biocompatible ceramic or metallic material, like titanium. Liketitanium, the selected material should also preferably be subject todetection within the host tissue by fluoroscopy, x-ray, and the like.

The specific dimensions of the hoop-like housing 11 may vary accordingto its intended use and the volume of cells 12 it contains.

In one preferred embodiment, the side wall of the first hoop element 14is about 0.055 inch in height and has an outer diameter of about 0.375inch. The open area has an inner diameter of about 0.325 inch where itjoins the inner edge of the chamfer 30 of the second central opening 24.The second central opening 24 has an inner diameter of about 0.326 incharound the outer edge of the chamfer 30. The first central opening 14has an inner diameter of about 0.275 inch and a depth of about 0.015inch, where it joins the interior ledge 26.

In this embodiment, the associated second hoop element 16 has a heightof about 0.025 inch; an outer diameter of about 0.326; and an innerdiameter (for its central opening 28) of about 0.250 inch. The range ofinterference necessary to snugly join the second hoop element 16 withinthe first hoop element 14 will of course depend upon the nature of thematerials selected.

The chamber includes a first porous wall element 32, a second porouswall element 34, and a sealing gasket or ring 36 that is sandwichedbetween them. The sealing ring 36 is made of a mesh polyester material.

The wall elements 32 and 34 and sealing ring 36 are sized to fit snuglywithin the confines of the hoop-like housing 11. And, as will bedescribed in greater detail later, at least one (and preferably both)porous wall elements 32 and 34 have certain physical characteristicsselected to protect and sustain the viability of the cells 12 within thehost.

The ring 36 has a central open region 38. The open ring region 38,together with the overlying first and second porous wall elements 32 and34, create the chamber 18 to hold the implanted cells 12 (see FIG. 4).

In making the assembly 10 shown in FIGS. 1 to 4, the practitioner laysone wall element 32 onto the ledge 26 formed in the first hoop element14. The practitioner then lays the sealing ring 36 upon the wall element32. Next, the practitioner inserts the desired amount of cells 12 to beimplanted into the open region 38 of the ring 36. The amount ofimplanted cells 12 is sufficient to induce the expected therapeuticresponse in the host.

The practitioner next lays the other wall element 34 over the first wallelement 32, sealing ring 36, and inserted cells 12. To complete theassembly 10, the practitioner presses the second hoop element 16 throughthe second central opening 24 into pressing engagement against theadjacent wall element 34. This seals the periphery of the cell holdingchamber 18, which now snugly rests within the open area of the hoop-likehousing 11.

Once assembled, one wall element 32 rests against the interior ledge 26and is there exposed through the first central opening 22. The otherwall element 34 rests against the second hoop element 16 and is thereexposed through its central opening 28.

FIGS. 5 to 10 show another implant assembly 10' that embodies thefeatures of the invention. Like, the implant assembly 10 previouslydescribed, the assembly 10' includes a cell chamber 18' formed by firstand second porous wall elements 32' and 34' and an intermediate sealingring 36'.

Unlike the first described implant assembly 10, the assembly 10' doesnot rely upon the hoop-like housing 11 to hold and seal the chamber 18'.Instead, a preformed peripheral weld 40 bonds and seals the edges of theporous wall elements 32' and 34' to the interior ring 36'.

In making the assembly 10' shown in FIGS. 5 to 10, the practitioner laysthe sealing ring 36' upon one wall element 32' and inserts the desiredamount of cells 12 to be implanted into the open region 38' of the ring36' (see FIG. 5). The practitioner overlays the other wall element 34'(as FIG. 6 shows). The practitioner then forms the weld 40 to seal theperipheral edges of the first and second wall elements 32' and 34' tothe ring 36' (as FIG. 8 shows). The weld compresses the peripheral edgeof the assembly 10' together, as FIG. 9 shows.

The practitioner selects a sealing technique that does not damage thecells 12 within the chamber 18'. For example, the inventors find thatsonic welding can be used without damage to the inserted tissue cells.

In a preferred embodiment (using the laminated structure 72 made asshown in FIGS. 11 to 16, as will be described later), the practitioneruses a Branson sonic welder. The welder is operated at 40 Khz, with941AES actuator, 947 m power supply, and 91 C power controller. The hornamplitude is about 1.4 mils and is operated at a hold time of about 0.3seconds; a weld time of about 0.20 seconds; a pressure of about 50 PSI;a trigger force of about 20 pounds; and a down speed of about 1.25(machine setting).

These are typical operating ranges for making the sonic weld and canvary according to the materials used and degree of cell loading withinthe chamber.

The integral assembly 10' formed in this manner can be implanteddirectly within host tissue, without use of an exterior housing.

Preferably, as FIG. 8 shows, the assembly 10' includes an attached clip42 made of a material that can be detected within the host tissue byfluoroscopy, x-ray, and the like. In this way, the practitioner caneasily locate the assembly 10' within the host, if required.

Like the first described embodiment, the specific dimensions of theassembly 10' may vary according to its intended use. And, like the firstdescribed embodiment, at least one (and preferably both) porous wallelements 32' and 34' have certain physical characteristics selected toprotect and sustain the viability of the cells within the host.

Regardless of the assembly used, the practitioner surgically implants itin the soft tissue 44 of the host (see FIG. 17). During surgery, thepractitioner positions the assembly 10 so that the exposed first andsecond wall elements 32 and 34 rest close to the surrounding host tissue44. In FIGS. 17 to 21, assembly 10 also encompasses assembly 10'.

The first and second wall elements 32 and 34 thereby together form thedesired boundary 46 between the biological system of the host tissue 44living outside the chamber 18 and the biological system of the implanttissue cells 12 living within the chamber 18.

For a period of time after implantation, the region of the host tissue44 immediately surrounding the implant assembly 10 is ischemic (see FIG.18). The region is ischemic, because the host treats the assembly 10 asa foreign body.

The host forms a wound area 48 around the assembly 10 (see FIG. 18). Thewound area 48 has spaces that become filled with wound exudate 50. Thewound exudate 50 keeps this area 48 ischemic.

Soon after implantation, host inflammatory cells enter and occupy theexudate area 48. "Inflammatory cells" include macrophages, foreign bodygiant cells, and fibroblasts.

The inflammatory cells try to remove the foreign implant assembly.Macrophages from the host try to ingest the foreign implant assembly 10.In some cases, the macrophages coalesce to form multinucleated giantcells. Fibroblast layers form to create a fibrous sac of cells andcollagen around the foreign implant assembly 10, commonly called theforeign body capsule 52 (see FIG. 20).

The inventors have discovered that it is not the foreign body capsule 52that most threatens the viability of the implanted cells during theischemic period. Rather, the existence of the cells is most threatenedduring the ischemic period when the boundary 46 itself fails to allowenough extracellular nutrients like glucose and other metabolic supportcompounds present at the boundary 46 to pass to the cells. Withoutmetabolic support, the implanted cells become dysfunctional or perish.

As FIG. 18 shows, the wound exudate 50 forms a fluid barrier between thevascular system of the host and the boundary 46. This barrier hindersthe extracellular passage of nutrients from the host vascular system tothe boundary 46. The concentrations of nutrients decrease as theytransit the exudate barrier to reach the boundary 46.

The host's inflammatory cells that in time enter the wound exudateregion 50 also create a metabolic sink. These cells compete for andfurther extract more of the host's extracellular nutrients before theyreach the boundary.

If the host is stimulated to grow new vascular structures 54 close tothe boundary 46, host endothelial cells will also enter the region 48.These cells begin the crucial process of forming the new vascularstructures 54. Still, their presence further contributes to themetabolic sink effect. The host's endothelial cells further reduce theavailability of nutrients for the implanted cells.

The ischemic period will end, if enough neovascular structures 54 fromthe host grow within the exudate region 50 close to the boundary 46 ofthe assembly 10 (as FIGS. 19 and 20 show). The close vascular structures54 shorten the extracellular path that nutrients must travel to reachthe boundary 46. The close vascular structures 54 provide nutrients inhigher concentrations to the implanted cells. Close vascularization alsotransports the therapeutic products generated by the implanted cells 12to the host.

However, all these desired benefits accrue only if the implanted cells12 survive the critical ischemic period.

The inventors have discovered that the diminished concentrations ofnutrients present at the boundary 46, although significantly reduced bythe exudate barrier and metabolic sink effects, are still enough tosustain the implanted cells. This is true, even in the presence of aforeign body capsule.

Still, the cells will die, if the boundary 46 itself lacks the capacityto let enough of the remaining nutrients through to the cells at asufficiently high rate. The inventors refer to this capacity as themetabolic transit value.

The inventors have discovered that the boundary 46 itself can alsopresent another significant barrier to the passage of nutrients. Theadded barrier effect of the boundary 46 can further reduce the alreadydiminished concentration of nutrients, until there is essentiallynothing left to sustain the cells.

The series barriers to the extracellular passage of nutrients (the woundexudate 50, the boundary 46, and the metabolic sink effect) also inhibitthe reverse passage metabolic wastes from the implanted cells.

The inventors have discovered that two principal factors threaten thesurvival of the implanted cells during the ischemic period. The firstfactor (which is conventionally recognized) is the failure to isolatethe cells from the natural immune response of the host. The secondfactor (which is not conventionally recognized) is the undesirableadditional barrier effect of the boundary 4 that impedes the essentialflux of already scarce nutrients to the implanted cells before closevascularization fully develops. The same barrier effect impedes the fluxof metabolic waste products away from the implanted cells to the host.

If the boundary 46 does not support the ongoing metabolic processes ofthe implanted cells while isolating them from the immune response of thehost during the ischemic period, the implanted cells will not live longenough to derive the benefits of close vascularization, if it occurs.

According to this aspect of the invention, then, the porous boundary 46is characterized in terms of its pore size; its ultimate physicalstrength; and its metabolic transit value. The first two characteristicsserve to isolate the implant tissue cells from the immune response ofthe host. The last characteristic serves to transfer nutrients and wasteproducts in support of the metabolic processes of implanted cells duringthe ischemic period, before close vascularization occurs. The lastcharacteristic sustains the viability of the implanted cells during theischemic period, even as a foreign body capsule forms.

According to another aspect of the invention, the assembly also includesan angiogenic material. The presence of an angiogenic materialstimulates the neovascularization required close to the boundary 46 tobring an end to the ischemic period.

According to yet another aspect of the invention, the porous boundary 46includes an interface 47 with the host tissue that is characterized by aconformation that supports and fosters the growth of vascular structuresby the host close to the boundary 46.

Further details of the beneficial characteristics of the boundary 46 andits associated host interface 47 will now be individually described.

BOUNDARY PORE SIZE

The boundary 46 has a pore size sufficient to isolate the implant tissuecells from the immune response of the host.

As used in this Specification, "pore size" refers to the maximum poresize of the material. The practitioner determines pore size usingconventional bubble point methodology, as described in PharmaceuticalTechnology, May 1983, pages 36 to 42.

As a threshold requirement, the pore size selected must make theboundary 46 impermeable to the vascular structure that forms close tothe boundary 46. Penetration of the pores by the vascular structurebreaches the integrity of the boundary 46, exposing the implanted cellsto the complete immune response of the host. Generally speaking, poresizes less than about 2 microns will block the ingress of vascularstructures.

The ultimate pore size selected also depends upon the species of thehost and the biologic relationship between the host and the donor of theimplant tissue cells.

When the implanted cells are from another animal species (i.e.,xenografts), the pore size must be sufficient to prevent the passage ofboth inflammatory cells and molecular immunogenic factors from the hostinto the implant tissue chamber. As used in this Specification,"molecular immunogenic factors" refers to molecules such as antibodiesand complement.

Pore sizes sufficient to block passage of both inflammatory cells andmolecular immunogenic factors in humans lie in the range of about 0.015micron. Of course, these pore sizes are also impermeable to vascularstructures.

When the implanted cells are from the same animal species but having adifferent genetic make up (i.e, allografts), the pore size usually mustbe sufficient to prevent the passage of only inflammatory cells from thehost into the implant cell chamber. In allografts, molecular immunogenicfactors do not seem to adversely affect the viability of the implantedcells. Still, some degree of tissue matching may be required forcomplete protection.

Pore sizes sufficient to block passage of inflammatory cells in humanslie in the range of below about 0.8 micron. These pore sizes, too, areimpermeable to vascular structures.

When the implanted cells are isografts (autologous implants ofgenetically engineered cells), the pore size must be sufficient only toprevent the isografts from entering the host. Still, with isografts, thepore size selected must also prevent ingress of vascular structures.

BOUNDARY STRENGTH

The boundary 46 has an ultimate strength value that is sufficient towithstand, without rupture, the growth of new vascular structures, thegrowth of new cells within the chamber 18/18', and other physiologicalstresses close to the host tissue. Keeping the boundary 46 secureassures isolation of the implanted cells from both the immunogenicfactors and inflammatory cells of the host.

These physiological stresses are caused when the host moves about incarrying out its normal life functions. The proliferation of implantedcells and the growth of vascular structures 54 also contributes to thephysiological stresses close to the boundary 46. The stresses challengethe physical integrity of the boundary 46 by stretching or otherwisedeforming it.

Absent a sufficient ultimate strength value, normal physiologicalstresses can rupture the boundary 46, exposing the implanted cells tothe full effect of the host's immune and inflammatory systems.

The inventors presently believe that ultimate strength values sufficientto withstand physiological stresses close to the host tissue withoutrupture in animals lie above about 100 pounds per square inch (PSI). Incomparison, the ultimate strength value for PVA hydrogel microcapsulesis only about 2 to 2.5 PSI.

The ultimate strength values are determined by measuring the tensilestrength of the material. Tensile strength is measured by ASTM D-412.

METABOLIC TRANSIT VALUE

The boundary 46 also has a metabolic transit value that sustains a fluxof nutrients into the chamber 18 and waste products from the chamber 18sufficient to sustain the viability of the implanted cells during theischemic period.

The metabolic transit value takes into account the permeability value(P) and the porosity value (PORE) of the boundary 46.

The Permeability Value

The permeability value (P) is the measure of the amount of solute thattravels through the boundary per unit time and unit surface area, givensome fixed external solute concentration (measured in cm/sec in thisspecification). Example 1 sets forth a methodology for determining thepermeability value according to this aspect of the invention.

The Porosity Value

The porosity value (PORE) represents the space in the boundary 46 thatdoes not contain material, or is empty, or is composed of pores.Expressed as a percentage, the porosity value (PORE) measures the %volume of the boundary 46 that is not occupied by boundary material.

To derive the porosity value PORE (in %) for materials having a POREequal to or greater than 10%, the practitioner uses the followingformula:

    PORE=100(1-(ρ.sub.b /ρ.sub.m)

where;

ρ_(b) is the density of the boundary as determined from its weight andvolume, and

ρ_(m) is the density of the boundary material.

To derive the porosity value PORE (in %) for materials having a POREless than 10%, the practitioner uses using a scanning electronmicroscope to obtain the number of pores and their average diameter onthe boundary. PORE is then derived according to the following formula:

    PORE=Nπ(d.sup.2 /4)

where:

N is the pore density and equals (p_(n) /a),

p_(n) is the number of pores in the boundary,

a is the total area of the boundary (in cm²), and

π is the transcendental constant 3.1416 . . . ,

d is the average diameter of the pores (in cm).

The inventors have found that, above a threshold minimum porosity value,the permeability value is the principal influence upon the overallmetabolic transit value. Still, below the threshold minimum porosityvalue, the metabolic transit value must also take into account theporosity value and the physical structure of the porous boundary 46.These considerations will be discussed later in greater detail.

To simplify the selection of an boundary 46, the inventors recommend theuse of boundaries having a porosity value (PORE) greater than theobserved minimum threshold value. Then, metabolic transit value and thepermeability value can be treated as the same.

As the following Example 1 shows, the inventors have discovered thatthere is a direct correlation between the metabolic transit value andimplanted cell survival during the ischemic period.

EXAMPLE 1

Embryonic lungs enclosed in membrane chambers having differentpermeability values were implanted in subcutaneous sites in rats.

1. Permeability

The permeability values for the membrane chambers were obtained forinsulin diffusion in a conventional benchtop diffusion chamber, made byCrown Glass Company, Somerville, N.J. (Part Number DC-100), usingradioactively labeled (¹²⁵ I) insulin as the solute (obtained from ICNBiochemicals) (a technique that is well known as steady-state diffusionin a diaphragm cell). The diffusion chamber had two chambers (which wibe called Chambers A and B), each with a volume of 3 ml. The diffusionchamber presented a membrane surface area between the two chambers(where diffusion occurs) of 0.7 cm².

The practitioner cuts the membrane material to be tested to apredetermined, known size.

If the membrane is hydrophobic, the practitioner wets the membranebefore conducting the permeability test, using conventional wettingtechniques.

The practitioner places the membrane in the diffusion chamber. Theassembly of the diffusion chamber locates the membrane between the twochambers of equal volume, called Chamber A and Chamber B. In this way,the practitioner also fixes the cross sectional area (A) of themembrane. The diffusion chamber is uniformly heated to a temperature ofabout 37 degrees C. during the test.

The practitioner loads equal amounts of buffer solution into Chamber Aand Chamber B. The buffer solution can vary. In this Example, thepractitioner can use phosphate buffered saline, 0.5% BSA as the buffersolution.

The practitioner then loads equal amounts of unlabeled (non-radioactive)insulin (about 3.4 micro units/ml) into Chamber A and Chamber B. Porcinepancreas insulin purchased from Sigma with an activity of 26.1 units/ml,or comparable material, can be used. The unlabeled insulin occupies anyadsorption sites that may be present.

The practitioner uniformly stirs the fluids within the chamber at about600 RPM, using a magnetic stir plate and magnetic stir rods (about 1 cmin length) placed in each Chamber A and B. The practitioner allows thesystem to equilibrate for about one hour.

The practitioner then removes a selected volume of buffer solution fromChamber A and adds back an equal volume of radioactive insulin. Theradioactive insulin suspension is filtered before use to remove free ¹²⁵I.

While stirring the fluids within Chamber A and Chamber B, thepractitioner draws equal aliquots of fluid from each Chamber A and B(e.g. about 15 uL) at 2, 4, 6, 8, 10, 15, and 30 minute intervals.

The practitioner then counts the radioactivity levels in the samplesusing a gamma counter.

The practitioner determines the change in the counts (i.e., insulinconcentration) in Chambers A and B per unit of time, suitably correctedfor background noise.

The practitioner graphs the count and time pairs for each Chamber interms of time versus the counts (with the counts being the Y-coordinatesand time being the X-coordinates), restricting the analysis to pointsfor which the counts in Chamber B are less than about 10% of the initialcounts in Chamber A. The practitioner then derives a linear equation,fitting the range of counts (y) over the set of times (x) for eachChamber according to the following equations:

For Chamber A:

    Y.sub.a =Y.sub.Intercept -(N.sub.a *X)

where

Y_(Intercept) is the count value where the graph intersects the Y axis,and

N_(a) is the slope of the Chamber A graph.

For Chamber B:

    Y.sub.b =Y.sub.Intercept =(N.sub.b *X)

where

Y_(Intercept) is the count value where the graph intersects the Y axis,and

N_(b) is the slope of the Chamber B graph.

The practitioner preferably uses a commercially available computerprogram to simplify the derivation process described above.

The practitioner then derives the permeability value (P) according tothe general expression: ##EQU1## where V_(b) is the volume of Chamber B

dM_(b) /dT is the change in counts in Chamber B per unit time, which isthe slope of the B graph derived above (N_(b)),

P is the permeability value,

A is the area of the boundary tested, and

M_(a) -M_(b) is the mass gradient of insulin across the membrane.

The practitioner knows V_(b) and A, which remain constant throughout thetest. The practitioner also knows dM_(b) /dT the slope of the graph forChamber B (N_(b)) from the linear equation derived for Chamber B. Thepractitioner converts the units of N_(b) (counts per min/min) intocounts per minute/sec by dividing by 60 (the number of seconds in aminute).

The practitioner calculates M_(a) by solving the linear equation derivedfor Chamber A for y when t=15 minutes (i.e., the mid point time for thetest). By using the mid point time for the test, the practitionerobtains an average value for the period of the test. The practitionersimilarly calculates M_(b) by solving the first order linear equationderived for Chamber B for y when t=15 minutes. From these values, thepractitioner calculates M_(a) -M_(b).

The practitioner can now derive the permeability value (in cm/sec) asfollows: ##EQU2##

Actually, the permeability value derived also includes the boundarylayer effects that are associated with inevitable stagnate fluid layersat the membrane surface in Chambers A and B during the test. To arriveat the "true" intrinsic permeability value for the boundary, thepractitioner would have to adjust for the boundary layer effects.However, for the purposes of this invention, a knowledge of the inherentmembrane permeability is not essential, because it will be proportionalto the experimental permeability value determined following themethodology detailed above.

Yet, the practitioner can follow the foregoing methodology to quantifythe relative permeability values for selected boundaries, since boundarylayer effects will remain constant as long as the stirring method usedremains the same.

The disclosed methodology can be used to assess whether a given boundaryfits the criteria established for the permeability value according tothis aspect of the invention.

2. Porosity

The porosity values (PORE) of the boundaries tested ranged from lessthan about 15% to greater than about 70%.

3. Determining Cell Survival

Embryonic lungs were removed from Lewis rat embryos between days 13.5and 17.5 of development. The lungs were kept on ice in Dulbecco'sModified Eagle's Medium (DMEM), 20% fetal bovine serum. The lungs wereminced until they were approximately 1 mm². Minced lung tissue (5-10 μl)was placed into implant assemblies like those shown in FIGS. 1 to 4. Thelung tissue was encapsulated within test membranes having variouspermeabilities, porosities, and pore sizes. The implant assemblies wereplaced in DMEM (20% fetal bovine serum) at 37 degrees C. until surgery,which occurred within 2 hours. The implant assemblies were implanted insubcutaneous or epididymal fat sites in male Lewis rats for 3 weeks.

After three weeks of implantation, the assemblies were explanted,trimmed of excess fat, and fixed with 2% glutaraldehyde in Sorensen'sbuffer. Sections of the assemblies were stained with hematoxylin andeosin.

Cell survival was scored based upon histological appearance of theimplanted cells. Tissues were scored as "excellent" if they had normalcharacteristics of lung tissue, such as epithelial tubules, cilia, andformed cartilage. Tissues were scored as "good" if the tissue were stillalive, but not well differentiated (for example, a high number ofmesenchymal cells). The tissues were scored as "poor" if no or few cellsremained alive.

In other histology studies using implanted pancreatic cells, survivalassessment would involve analyzing the differentiated function of thepancreatic cells in terms of their insulin release in the response to aglucose challenge.

Table 1 shows the permeability value for those boundaries having aporosity value (PORE) greater than 70%, correlated with the survival ofthe implanted lung tissues.

                  TABLE 1                                                         ______________________________________                                        Membranes with PORE > 15%                                                                  Pore Size or MW                                                                             Perme-  Tissue                                     Membrane     Cutoff        ability*                                                                              Survival                                   ______________________________________                                        cellulose acetate.sup.1                                                                    unknown       9       excellent                                  cellulose acetate.sup.1                                                                    unknown       5.3     excellent                                  BIOPORE ™.sup.2                                                                         0.45 μm    2.6     excellent                                  polyvinyl difluoride.sup.1                                                                 unknown       2.5     good                                       cellulose mixed                                                                             1.2 μm    2.0     poor                                       ester.sup.2                                                                   polyvinyl difluoride.sup.1                                                                 unknown       1.7     good                                       polypropylene.sup.3                                                                        0.075 μm   1.4     poor                                       cellulose acetate.sup.1                                                                    unknown       1.3     poor                                       cellulose mixed                                                                            0.45 μm    0.9     poor                                       ester.sup.2                                                                   polyethylene.sup.3                                                                         0.08 μm    0.9     poor                                       cellulose.sup.4                                                                            300 kD        0.6     poor                                       cellulose.sup.4                                                                             50 kD        0.2     poor                                       ______________________________________                                         *× 10.sup.-4 cm/s                                                       .sup.1 Baxter Healthcare Corporation (Deerfield, Il)                          .sup.2 Millipore Corporation (Bedford, Ma)                                    .sup.3 Hoechst Celanese (Charlotte, NC)                                       .sup.4 Spectrum Medical Instruments (Los Angeles, Ca)                    

Table 2 shows the permeability value of those boundaries having aporosity value (PORE) less than 15%, correlated with the survival of theimplanted cells.

                  TABLE 2                                                         ______________________________________                                        Membranes with PORE < 15%                                                                             Perme-   Tissue                                       Membrane*   Pore Size   ability  Survival                                     ______________________________________                                        NUCLEPORE.sup.1                                                                           0.8         4.4      Fair                                         NUCLEPORE   0.4         3.1      Poor                                         NUCLEPORE   0.22        2.3      Poor                                         PORETICS.sup.2                                                                            0.1         2.2      Poor                                         PORETICS    0.08        0.5      Poor                                         PORETICS    0.05        1.2      Poor                                         PORETICS    0.03        0.9      Poor                                         PORETICS    0.01        0.2      Poor                                         ______________________________________                                         *polycarbonate                                                                  × 10.sup.-4 cm/s                                                      .sup.1 Nuclepore Corporation (Pleasanton, Ca)                                 .sup.2 Poretic Corporation (Livermore, Ca)                               

Tables 1 and 2 demonstrate the direct relationship between the metabolictransit value of the boundary and implanted cell survival. Moreparticularly, the Tables show that implanted cell survival significantlyimproves when the permeability value of the boundary increases.

For the type of cells studied in Example 1, boundaries having apermeability value for insulin less than about 1.5×10⁻⁴ cm/sec, asdetermined using the described methodology, consistently did not supportcell survival, regardless of the porosity value. Yet, boundaries havinga permeability value for insulin greater than about 1.5×10⁻⁴ cm/sec anda porosity value greater than about 15% uniformly supported vigorouscell survival.

Boundaries having a lower porosity value (less than about 15%) alsosupported cell survival (see Table 2). Still, the metabolic transitvalue for these less porous boundaries requires a higher relativepermeability value. For the type of cells studied in Example 1,boundaries having a lower porosity value (less than about 15%) supportedcell survival when the permeability value for insulin was greater thanabout 4.0×10⁻⁴ cm/sec.

The inventors believe that, when considering less porous boundaries,their specific physical structure must also be taken into account. Theless porous interfaces used in Example 1 were track-etched membranes.These membranes have uniform cylindrical pores separated by relativelylarge, nonporous regions.

The poor tissue survival using the low porosity boundaries could be dueto uneven localization of areas of high permeability, or due toconstraints produced by cells on the particular physical properties ofthe track-etched membranes. For example, the cells may be more efficientat plugging up the cylindrical pores of the track-etched membraneseither with cell extensions or cell secretions. Thus, although thetrack-etched membranes have high permeability values in vitro, theresponse of the cells in vivo may prevent the attainment of sufficientmetabolic transit to support the graft cells.

Example 1 demonstrates a methodology that can be followed to identifyfor other cell types the applicable metabolic transit value that assurescell survival during the ischemic period after implantation.

The absolute permeability and porosity values that constitute a givenmetabolic transport value will depend upon the type of cell and themethodologies of determining permeability and porosity. Differentconditions will give different absolute values. Still, regardless of thetest conditions, the relative differences in permeability and porosityvalues derived under constant, stated conditions will serve as anindicator of the relative capabilities of the boundaries to supportimplanted cell viability.

Tables 1 and 2 also show that good tissue survival occurs even withmembrane materials that are subject to the formation of an avascularfibrotic response (the so-called "foreign body capsule"). The fact thatthese membrane materials create this response has, in the past, led tothe widely held view that the formation of the foreign body capsulecaused poor diffusion of nutrients. Example 1 shows the error of thisconventional wisdom.

As Table 1 shows, the use of relative thicker cellulose acetatemembranes with 0.45 micron pore size (130 microns thick) having aninsulin permeability of 0.9×10⁻⁴ cm/sec results in poor tissue survival.On the other hand, the use of relatively thinner cellulose acetatemembranes with the same approximate pore size (10 microns thick) andhaving a greater permeability of 5.3×10⁻⁴ cm/sec results in excellenttissue survival.

The thickness of the membrane does not alter the foreign body response;a foreign body capsule will form whether the membrane is relativelythick or thin. However, membrane thickness does alter the permeabilityvalue.

Thus, the cells died when the thicker boundary was used, not because ofthe formation of the foreign body capsule, but because of poor nutritionand poor waste removal due to the low permeability of the thickerboundary. The tissue survived when the thinner boundary is used, becausethe higher permeability provided improved cell nutrition and improvedwaste removal to support cell metabolism, even when the same foreignbody capsule forms.

EXAMPLE 2

In an experiment, the practitioner grew RAT-2 fibroblasts (ATCC CRL1764) in 20% Fetal Bovine Serum, 2 mM l-glutamine, and DMEM (Sigma)(high glucose) until 100% confluent. The RAT-2 cells were split 1:2 inthe above media, 16 to 24 hours before surgery.

On the day of surgery, the cells were washed with 15 ml of HBSS (noions) and trypsinized off the culture flask. The practitionerneutralized the trypsin by adding 5 ml of the above media. Thepractitioner pelleted the cells by centrifugation (1000 rpm, 10 minutes,at 22 degrees C.).

The pelleted cells were counted and resuspended in media in threeconcentrations: 5.3×10³ cells/10 μl; 5.8×10⁵ cells/10 μl; and 5.8×10⁶cells/10 μl.

Implant assemblies like that shown in FIGS. 1 to 4 having boundaries ofdiffering permeability values were made. The permeability values rangedfrom 0.2×10⁻⁴ cm/sec to 9×10⁻⁴ cm/sec (see Tables 1 and 2 to follow).The total boundary area for each assembly was about 0.77 cm².

The various cell concentrations were loaded into the assemblies. Thepractitioner implanted the assemblies both subcutaneously and within theepididymal fatpad of host rats.

After 3 weeks, the assemblies were explanted and examinedhistologically, as described previously.

The inventors observed that assemblies loaded with 5.8×10³ cells and5.8×10⁵ cells displayed excellent results, given sufficient boundarypermeability values. After 3 weeks of implantation, the initial load of5.8×10⁵ cells proliferated to approximately 2.0×10⁷ cells. The inventorsobserved that assemblies having higher initial loads of 5.8×10⁶ cellsdisplayed poorer results.

Lower initial loads (less than 5×10⁶) were able to survive the ischemicperiod and even proliferate 30 to 3000 fold. The final cell counts inthe assemblies with lower initial loads were three times higher than theinitial load of the assemblies that failed because of higher initialloads. Thus, high loads of cells (greater than 5×10⁶) are unable tosurvive during the ischemic period, yet the same cell loads are able tosurvive after the ischemic period as progeny of the cells from lowerinitial loads.

Close Vascularization at the Boundary

(1) Presence of Angiogenic Material

Neovascularization close to the boundary is essential to the long termsurvival of the implanted cells within the host. The inventors havefound that the host will not grow new vascular structures 54 close tothe boundary (as FIGS. 24 and 25 show), unless it is stimulated to doso. Without proper stimulation, the ischemic period never ends, becausea classical foreign body reaction occurs.

The assembly 10 therefore includes an angiogenic material 56 forstimulating neovascularization close to the boundary.

The specific identity of the angiogenic material 56 is not known. Still,the inventors have determined that the presence of certain cellsstimulate neovascularization, while others do not.

For example, the presence of lung tissues; pancreatic islets; adultpancreatic ducts; and cultured cell lines of fibroblasts, mammary gland,and smooth muscle cells induces or stimulates neovascularization, whencompared to the vascularization on control grafts where these cell typeswere not present.

On the other hand, the presence of primary skin fibroblasts andmicrovascular endothelial cells do not induce neovascularization.

The inventors believe that certain cells induce or stimulateneovascularization by secreting angiogenic factors. Because the stimuluscrosses membranes that are impermeable to cells, it must be a molecularsignal that the living cell generates. This further underscores the needto support the implanted cells during the ischemic period. If angiogenicsource cells perish, the molecular signal stops, and theneovascularization process comes to a halt.

According to this aspect of the invention, when cells are implanted thathave a desired therapeutic effect, but do not secrete angiogenicmaterial, the assembly 10 includes a separate angiogenic source cell ormaterial 56.

Following the invention, the practitioner selects an boundary 46 havinga sufficient metabolic transit value to support the viability of theimplanted cells, i.e., the angiogenic source cells and othernon-angiogenic, therapeutic cells (when present) implanted with them.The practitioner also selects a pore size and ultimate physical strengthto make the boundary 46 impermeable to the neovascular growth that theangiogenic source cells stimulate.

Alternatively, the practitioner may coat the exterior of the boundary 46itself with an angiogenic material 56. Of course, the coated boundary 46still must have sufficient pore size, ultimate strength, and metabolictransit value to sustain the cells 12 isolated behind the boundary 46.

Because the new vascular structures 54 cannot penetrate the boundary 46,and because the angiogenic signal to the host continues, the newvasculature proliferates close to the boundary 46.

As FIG. 21 shows, when the cells 12 die during the ischemic period, andclose vascularization is not stimulated, the fibroblasts of the foreignbody capsule 52 become closely packed and dense. However, as FIG. 20shows, when the cells 12 survive the ischemic period, and the process ofclose vascularization is stimulated, the fibroblasts of the foreign bodycapsule 52 is altered to form a less dense and more dispersed structure.

(2) Conformation for Close Vascularization

In the preferred embodiment, the porous boundary 46 includes aninterface 47 with the host tissue that is characterized by a structuralconformation that further enhances the growth of vascular structures bythe host close to the boundary.

To achieve this result, each wall element 32/32' and 34/34' of theassemblies 10/10' includes a first porous region 58 and a differentsecond porous region 60. The first porous region 58 comprises theboundary 46 previously described. The second porous region 60 comprisesthe interface 47.

The first porous region 58 faces the implanted cells 12 (see FIG. 20).The first porous region 58 has the boundary characteristics, abovedescribed, of pore size; ultimate physical strength; and metabolictransit value. It is this region 58 that isolates the implanted cellsfrom the immune mechanisms of the host, while sustaining their viabilitythrough the flux of nutrients and wastes during the ischemic period.

The second porous region 60 faces the host tissue 44 and forms theinterface 47 with it (see FIG. 20). The second porous region 60 has anarchitecture that enhances the formation of vascular structures 54 closeto the boundary 46. The formation of these vascular structures 54 withinthe second region 60 mark the end of the ischemic period.Vascularization in the second region 60 sustains the viability of theimplanted cells 12 after the ischemic period ends.

A foreign body capsule 52 still forms about the implanted assembly 10.However, close vascularization within the second porous region 60 canalter the normal configuration of the foreign body capsule 52. As FIG.20 shows, a life sustaining vascular bed forms within the capsule 52close to the boundary 46, keeping flattened macrophages, foreign bodygiant cells, and fibroblasts from pressing against and blocking theboundary 46.

Because of the pore size, strength, and permeability characteristics ofthe porous first region 58, it is impermeable to the neovasculature 54formed in the second region 60.

The inventors believe that close vascularization occurs if the threedimensional conformation of second region 60 creates certain hostinflammatory cell behavior.

The inventors have observed by light and electron microscopy that closevascularization occurs if, in the initial period of implantation, atleast some macrophages entering the material are not activated.Activated macrophage are characterized by cell flattening.

The inventors observe close vascularization in regions of an implantwhere the macrophages that have entered the cavities of the materialretain a rounded appearance when viewed through light microscopy(˜400×). At 3000× (TEM) the rounded macrophage is observed to havesubstantially conformed to the contours of the material. Although thereis a correlation with macrophage shape, it is not clear that macrophagescontrol the observed response. However, it is clear that invasion of thestructure by host cells is required. Although the bulk of the cellsappear to be macrophages, it is possible that other inflammatory cellscontrol the response, therefore the inventors refer to the invadingcells as "inflammatory cells," which include but are not limited tomacrophages.

On the other hand, foreign body capsule formation occurs when, in theinitial period of implantation, inflammatory cells in contact with theimplant material flatten against those portions of the material whichpresent an area amenable to such flattening behavior by an inflammatorycell.

The material for the second region 60 that results in formation of closevascular structures is a polymer membrane having an average nominal poresize of approximately 0.6 to about 20 μm, using conventional methods fordetermination of pore size in the trade. Preferably, at leastapproximately 50% of the pores of the membrane have an average size ofapproximately 0.6 to about 20 μm.

The structural elements which provide the three dimensional conformationmay include fibers, strands, globules, cones or rods of amorphous oruniform geometry which are smooth or rough. These elements, referred togenerally as "strands," have in general one dimension larger than theother two and the smaller dimensions do not exceed five microns.

In one arrangement, the material consists of strands that define"apertures" formed by a frame of the interconnected strands. Theapertures have an average size of no more than about 20 μm in any butthe longest dimension. The apertures of the material form a framework ofinterconnected apertures, defining "cavities" that are no greater thanan average of about 20 μm in any but the longest dimension.

In this arrangement, the material for the second region has at leastsome apertures having a sufficient size to allow at least some vascularstructures to be created within the cavities. At least some of theseapertures, while allowing vascular structures to form within thecavities, prevent connective tissue from forming therein because of sizerestrictions.

Further details of the material are set forth in copending U.S.application Ser. No. 735,401 entitled "Close Vascularization ImplantMaterial" filed Jul. 24, 1991, which is incorporated into thisSpecification by reference.

Making a Boundary

FIGS. 11 to 16 show a method of making a preferred embodiment of thewall elements 32 and 34 that forms the boundary. The method integrallyjoins material selected for the first region 58 to another materialselected for the second region 60. The two joined materials form thecomposite, or laminated, structure 72 shared by both wall elements 32and 34. The laminated structure 72 joins the interface 47 to theboundary 46.

In the illustrated embodiment, a porous PTFE membrane material having athickness of about 35 microns and a pore size of about 0.4 micron isselected for the first region 58. This material is commerciallyavailable from Millipore Corporation under the tradename BIOPORE™.

The porous material selected for the first region 58 has a thickness ofabout 30 microns and an ultimate (tensile) strength value of at least3700 PSI, which is well above the desired minimum value. The selectedmaterial has pore size of 0.35 microns, which blocks the passage ofinflammatory cells. The selected material has a permeability value forinsulin of 2.6×10⁻⁴ cm/sec and a porosity value of greater than 70%. Themembrane therefore meets the metabolic transit value requirements.

It should be appreciated that other, comparable materials can meet thestated requirements for the first region 58. For example, polyethylene,polypropylene, cellulose acetate, cellulose nitrate, polycarbonate,polyester, nylon, and polysulfone materials can be used. Mixed esters ofcellulose, polyvinylidene, difluoride, silicone, and ployacrylonitrilecan also be used.

In the illustrated embodiment, a membrane material made by W. L. Goreand Associates (Elkton, Md.) under the tradename GORE-TEX™ is selectedfor the second region 60. The GORE-TEX™ material comprises a microporousmembrane made from PTFE. The membrane is 15 microns thick and has a poresize of 5 microns. Polyester strands 61 join the PTFE membrane to form abacking for it. The backing has a depth of about 120 microns.

The GORE-TEX™ material also has an ultimate strength value well abovethe desired minimum value. The conformation of the polyester strands 61also meets the criteria, set forth earlier, for promoting the growth ofneovascular structures.

In Step 1 (see FIGS. 10 and 11) the practitioner secures the edges of astrip of the Gore-Tex material (second region 60) to a lamination slide62, with the polyester backing 61 facing the slide 62.

In Step 2 (see FIG. 13), the practitioner places 2 or 3 laminationslides 62 side-by-side on a work surface. Using a syringe 64, thepractitioner applies cement or adhesive in continuous filaments 66 in aback and forth pattern across the lamination slides 62. The practitionertouches the syringe tip 64 to the work surface at the end of eachfilament 66 to begin a new filament 66.

Step 2 forms a criss-crossing pattern of cement filaments 66 across thesecured strips of the second region material, as FIG. 13 shows.

The cement selected can vary. For example, the cement can be celluloseacetate or similar epoxy material. In the illustrated embodiment, thecement comprises a mixture of Vynathene EY 90500 ethylene vinyl acetatecopolymer (EVA) resin and toluene (made by Mallinckrodt).

In forming the EVA cement mixture, the practitioner adds about 30 gramsof resin and an equal amount of toluene to a bottle. The practitionerseals the bottle to allow the resin to dissolve. The bottle may beperiodically shaken to accelerate this process.

The relative amounts of resin and toluene may have to be slightlyadjusted to arrive at the right consistency for the cement. If thecement is too thin to form continuous filaments when applied, use lesstoluene. If the cement is to viscous to be expressed from the syringe,use more toluene. Small changes in the amount of toluene added result issignificant changes in the viscosity of the cement.

In Step 3 (as FIG. 14 shows), the practitioner places preformed stripsof the BIOPORE™ membrane material (first region 58) upon the cementfilaments 66 applied in Step 2. In the illustrated embodiment, thepractitioner precuts the Biopore™ membrane material into disks havingthe diameter desired for the wall elements 32 and 34.

In Step 4 (as FIG. 15 shows), the practitioner lays a strip of releasematerial 68 (like Patapar) over the first region material 58 and coversthe layered structure with another lamination slide 70. The practitionerclamps the lamination slides 62 and 70 together, bringing the membranelayers into intimate contact.

In Step 5, the practitioner places the clamped lamination slides 62 and70 in an oven for about 5 to 10 minutes at a temperature of about 80degrees C. The heat melts the EVA cement.

In Step 6, the heated lamination slides 62 and 70 are allowed to cool toroom temperature. Upon cooling and solidification, the filaments 66securely join the BIOPORE™ membrane material to the Gore-Tex™ membranematerial. The practitioner then unclamps the lamination slides 62 and 70and removes the finished composite structure 72 (in strips).

In Step 7 (as FIG. 16 shows), the practitioner lays the compositestructure 72 strips on a polypropylene cutting slab 74. The practitioneraligns a presized punch 76 over each precut disk, striking the punchwith a hammer. The practitioner thereby frees the wall elements 32 or 34formed of the composite structure of the desired dimensions. Smallscissors may be used to snip any adherent polyester strands not cut bythe die.

Implant assemblies 10/10' are made using the wall elements in the mannerpreviously described.

It should be appreciated that the first region material 58 can beapplied to the second region material 60 by various alternative means toform the laminated structure 72. For example, the first region material58 can be extruded in place upon the second region material 60.

EXAMPLE 3

Assemblies like that shown in FIGS. 1 to 4 and constructed according tothe foregoing process have been successfully used to accomplish completecorrection of diabetes in partially pancreatectomized andstreptozotocin-treated rat hosts. The animals were corrected up to 293days. Upon removal of the implants, the animals reverted to a diabeticstate. Histology of the implants revealed the presence of vascularstructures close to the boundary.

These assemblies presented a boundary area of about 0.77 cm². Eachassembly sustained an initial cell load of about 600 pancreatic islets(or about 600,000 pancreatic cells).

When implanted, the assemblies sustained cell densities of about 200,000islets/cm³. These assemblies, made and used in accordance with theinvention, supported 8 times more pancreatic islets in a given volumethan the CytoTherapeutics assemblies (having cell densities of only25,000 islets/cm³).

Deriving a Therapeutic Loading Factor

As earlier described, one aspect of the invention provides the abilityto identify a metabolic transit value associated with a given cell type.Knowing the required metabolic transit value, in turn, makes it possibleto identify the clinically practical region of operation, where compactimplant assemblies can sustain therapeutically large volumes of cells.

This aspect of the invention provides the methodology to derive and usea therapeutic loading factor (L) to characterize and predict theclinical effectiveness of a given implant assembly for a given celltype.

The therapeutic loading factor (L) takes into account the number ofcells (N) that are required to be implanted to achieve the desiredtherapeutic effect; the effective area (A) of the boundary between theimplanted cells and host that the host can be reasonably expected totolerate; and the metabolic transit value (T) needed to sustain cellviability.

The therapeutic loading factor for a given implant assembly and givenimplanted cell type can be expressed as follows:

    L.sub.c =(A/N.sub.c)*T.sub.min

where

c is the given cell type,

L_(c) is the therapeutic loading factor for the given cell type,

A is the area of boundary between the implanted cells and the hostoffered by the given implant assembly,

N_(c) is the number of cells supported by the boundary area (A), and

T_(min) is the minimum metabolic transit value that will support cellsurvival during the ischemic period, determined according themethodology set forth in Example 1.

If the practitioner selects boundaries having a porosity value ofgreater than 15%, then the permeability value (P) alone can be used toexpress the metabolic transit value (T). The therapeutic load factor canthen be expressed:

    L.sub.c =(A/N.sub.c)*P.sub.min

where P_(min) is the minimum permeability value that will support cellsurvival during the ischemic period.

In the assemblies described in Example 3, the observed ratio between theboundary area and the number of implanted cells (A/N_(c)) for thesuccessful implantation of pancreatic cells was 128 μm² /pancreaticcell. The inventors believe that a somewhat larger ratio of about 150μm² /pancreatic cell will provide a satisfactory margin for varianceamong different hosts.

As earlier discussed, given a boundary porosity value of greater than15%, a permeability value (P) greater than about 1.5×10⁻⁴ cm/sec forinsulin should be provided a metabolic transit value that will sustaincell survival during the ischemic period and afterward.

FIG. 22 shows the therapeutic loading curve for pancreatic cellsgenerated based upon the above considerations. The curve displays thepredicted region of cell survival in terms of the boundary area-to-cellnumber ratio A/N (x-coordinate) and permeability value P (y-coordinate)(given a porosity value of greater than about 15%).

FIG. 22 predicts that assemblies operating to the right of thetherapeutic loading curve will sustain implanted pancreatic cells. FIG.22 predicts that assemblies operating to the left of the therapeuticloading curve will not.

The inventors believe that a human diabetic will require thetransplantation of about 250,000 pancreatic islets (or about 250 millionpancreatic cells) to derive a therapeutic benefit. With this in mind,one can calculate a range of sizes for an implant assembly based uponthe A/N ratio.

The equation for calculating the side dimension (L) in cm of a squareimplant assembly based upon the A/N ratio is as follows: ##EQU3## where:the factor 10⁻⁸ converts micron² to cm².

The equation for calculating the diameter (D) in cm of a round implantassembly based upon the A/N ratio is as follows: ##EQU4## where: thefactor 10⁻⁸ converts micron² to cm².

Table 3 lists a range of L's and D's at different A/N ratios for animplant assembly holding 250,000 pancreatic islets

    ______________________________________                                        A/N    A (cm.sup.2)/side                                                                              L(cm)   D(cm)                                         ______________________________________                                        128    160              12.6    14.3                                          150    188              13.7    15.5                                          200    250              15.8    17.8                                          328    410              20.2    22.8                                          463    579              24.0    24.1                                          ______________________________________                                    

Based upon the foregoing considerations, the inventors believe that A/Nratios less than about 200 μm² /pancreatic cell define the operatingregion of implant assemblies that offer compact, clinically practicalimplant boundary areas. FIG. 22 shows this preferred region.

As FIG. 22 also shows, a practitioner can provide an implant assemblythat combines the benefits of compact size with the ability to sustainthe requisite therapeutical number of cells, by selecting a permeabilityvalue for the boundary that achieves a region of operation to the rightof the therapeutic loading curve. The practitioner also selects theprescribed pore size and ultimate physical strength determined inaccordance with the invention.

FIG. 22 shows that the prior art hollow fiber implant assembly made byCytoTherapeutics (described in the background section of thisspecification) falls well outside the preferred region of clinicallypractical operation. This assembly offers an A/N ratio of about 328 μm²/pancreatic cell, about 1.5 times the A/N ratio of the invention.

FIG. 22 also shows a prior art hollow fiber implant assembly made by W.R. Grace and Co. (Lexington, Mass.), as reported by Proc. Natl. Acad.Sci. U.S.A., Vol. 88, pp. 11100-11104 (December 1991).

Each hollow fiber had a length of 2-3 cm, and an inside diameter of0.177 cm. There were 200 to 400 pancreatic islets loaded into each fiberfor implanation. Taking an average length of 2.5 cm and an average cellload of 300 islets, the associated A/N ratio is 463, more than twice theA/N ratio of the invention.

The foregoing establishes a methodology to derive and use a therapeuticloading factor (L) for pancreatic islets. This methodology can befollowed to identify a therapeutic loading factor for other cell typesand other ranges of metabolic transit values. The absolute value of thetherapeutic loading factor derived will of course depend upon the typeof cell and the methodologies used to determine permeability andporosity. Different conditions will give different absolute values forthe therapeutic loading factor.

Still, regardless of the test conditions, the relative differences inthe A/N ratios, permeability values, and porosity values derived underconstant, stated conditions will serve as a means to characterize andpredict the clinical effectiveness of a given implant assembly for agiven cell type.

The following claims further define the features and benefits of theinvention.

We claim:
 1. An assembly for embedding in host tissue, where it isexposed to physiological stresses and vascularization by host tissuecomprisingwall means defining a chamber for holding cells while embeddedin host tissue, the wall means including means for forming a porousboundary between host tissue and the cells in the chamber, the porousboundary being characterized bya tensile strength and a pore sizesufficient to isolate the implanted cells from an immune response ofhost tissue in which the chamber is embedded; and a metabolic transitvalue including a permeability value of greater than about 1.5×10⁻⁴cm/sec. for ¹²⁵ I-labeled insulin as determined by pseudo-steady statediffusion in a diaphragm cell, whereby the wall means sustains a flux ofnutrients from host tissue in which the chamber is embedded to the cellsin the chamber and waste products from the cells in the chamber to hosttissue in which the chamber is embedded in the absence of close vascularstructures to thereby sustain viability of the cells in the chamberuntil vascular structures of host tissue form close to the porousboundary.
 2. An assembly according to claim 1and further including anangiogenic material that, when embedded in host tissue, stimulates thegrowth of vascular structures by host tissue in which the chamber isembedded close to the boundary.
 3. An assembly according to claim 1 or2and further including a second material that overlies the porousboundary and forms an interface with host tissue in which the chamber isembedded, the second material having a structural conformation that,when embedded in host tissue, supports the growth of vascular structuresby host tissue close to the boundary.
 4. An assembly according to claim1 or 2wherein the pore size blocks the passage of molecular immunogenicfactors from host tissue to the cells in the chamber.
 5. An assemblyaccording to claim 1 or 2wherein the pore size blocks the passage ofinflammatory cells from host tissue to the cells in the chamber.
 6. Anassembly according to claim 1wherein the metabolic transit valueincludes a porosity value that is greater than about 15%, wherein theporosity value represents the volume in the porous boundary occupied bypores, expressed as a percentage of the total volume of the porousboundary.
 7. An assembly according to claim 1 or 2wherein the chambercontains a given number of cells (N), wherein the porous boundary coversa given area (A), and wherein A/N is less than about 200 μm² /cell. 8.An assembly according to claim 1 or 2wherein the tensile strength isgreater than about 100 pounds per square inch (PSI).
 9. A method ofimplanting cells comprising the steps ofsurrounding at least a portionof the implanted cells with a porous boundary that has a tensilestrength and a pore size sufficient to isolate the implanted cells froman immune response of host tissue; and a metabolic transit valueincluding a permeability value of greater than about 1.5×10⁻⁴ cm/sec.for ¹²⁵ I-labeled insulin as determined by pseudo-steady state diffusionin a diaphragm cell, and embedding the porous boundary within hosttissue whereby the porous boundary sustains a flux of nutrients fromhost tissue to the cells within the boundary and waste products from thecells within the boundary to host tissue in which the boundary isembedded sufficient to maintain viability of the cells within theboundary until vascular structures of host tissue form close to theboundary.
 10. A method according to claim 9 and further including thestep of exposing the boundary to an angiogenic substance that stimulatesthe formation of vascular structures by host tissue close to theboundary.
 11. A method according to claim 9 or 10 and further includingthe step of, before embedding the boundary in host tissue, overlaying onthe boundary a second material that has a structural conformation thatsupports the growth of vascular structures by host tissue close to theboundary.